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In the Communication on the following pages F. C. Simmel and co-workers describe a molecular device based on a DNA aptamer whichcan be instructed to repeatedly bind and release the protein thrombin.

3549Angew. Chem. Int. Ed. 2004, 43, 3549 DOI: 10.1002/anie.200353537 � 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

DNA Structures

ADNA-Based Machine That Can Cyclically Bindand Release Thrombin**

Wendy U. Dittmer, Andreas Reuter, andFriedrich C. Simmel*

Molecular machines can be used to produce controllednanoscale movements.[1] In particular, the unique molecularrecognition properties of DNA have been used to devise anumber of nanomechanical devices that can be reversiblyswitched between several distinct conformations.[2] Althoughthese devices display a variety of motions including rotationaland stretching-type movements,[3,4] they do not perform any

particular function. DNA structures with functionality can befound in DNA aptamers, which are short stretches of single-stranded DNA selected from a random pool of DNAsequences for their binding affinities to proteins or smallmolecules.[5] We report here on the construction of a protein-bearing molecular machine based on a DNA aptamer, whichcan be instructed to grab or release the human blood-clottingfactor, a-thrombin, depending on an operator DNA sequenceaddressing it. In the operation of this DNA nanomachine, thethrombin-binding DNA aptamer is mechanically switchedbetween a binding and a nonbinding form. The functioning ofthis “molecular hand” is demonstrated by gel electrophoresis,fluorescence resonance energy transfer (FRET), and fluores-cence anisotropy measurements.

The machine is based on the 15-base DNA sequence 5’-GGTTGGTGTGGTTGG-3’, which has been found to bind

strongly to a-thrombin[6] with a dissociation constant on theorder of 3–450 nm.[7] In the presence of potassium ions, theaptamer assumes a conformation characterized by twostacked guanine quadruplex structures (see Figure 1). Toconstruct the machine, we modified the aptamer at the 5’-endby adding a 12-base “toehold” section to address the device.The 5’-end was chosen in order not to interfere with thrombinbinding sites.[8] We validated, using melting-curve experi-ments monitoring the absorbance, that in the presence of K+

ions, the aptamer device (AP) indeed assumes its G quartet

Figure 1. Representation of the operation cycle of the aptamer-based molecular machine in the presence of thrombin (for an explanation see thetext). Sequences are shown for the labeled strands A3, Q5, and R.

[*] Dr. W. U. Dittmer, A. Reuter, Dr. F. C. SimmelSektion Physik and Center for NanoscienceLMU MunichGeschwister Scholl Platz 1, 80539 Munich (Germany)Fax: (+49)89-2180-3182E-mail: simmel@lmu.de

[**] We thank Prof. Dr. John S. McCaskill for helpful discussions andProf. Dr. J:rg P. Kotthaus for continuous support. This work wasfunded by the Deutsche Forschungsgemeinschaft, the BavarianState Ministry of Sciences, Research, and the Arts, and theAlexander von Humboldt Foundation.

Communications

3550 � 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/anie.200353537 Angew. Chem. Int. Ed. 2004, 43, 3550 –3553

form.[9] The operation cycle of the machine is depicted inFigure 1. Upon the addition of thrombin (TB), strand APbinds to the protein (I in Figure 1). By the addition of an“opening” strand Q, the DNA device extends to a duplexstructure AP-Q, which is not capable of binding to TB (III). Qis complementary to the toehold and the first ten bases of theaptamer sequence and additionally has an eight-base-longsequence at the 3’-end which serves as a point of attachmentfor the fully complementary “removal” strand R. This is thefundamental operating principle used to cycle DNAmachinesthrough their various conformations.[4] Q was not chosen to becompletely complementary to AP, because Rwould otherwisealso contain the full aptamer sequence and thus could bind tothrombin. Removal of Q by R returns the device to its originalconformation (I), where it binds to TB again.

We observed the operation of the machine with anelectrophoretic band-shift experiment (Figure 2). Binding of

AP (lane a) to thrombin results in a strong band shift (lane b).By the addition of Q, thrombin is released from AP and aband appears for the duplex AP–Q (lane c). Upon addition ofR, AP is released and can bind to thrombin once again(lane g). This can be judged from the reappearance of theAP–TB band and a second band from the “waste” productQ–R. An additional, faint band is observed in lanes b and gjust below that corresponding to AP–TB. It is found that theintensity of this band varies from one thrombin sample toanother. Furthermore, it is more intense with older thrombinsamples than with fresh ones. We therefore attribute thisadditional band to an impurity in our thrombin solution,possibly a thrombin decay product to which the aptamer canalso bind. In lane h, further addition of Q releases thrombinagain and two closely spaced bands appear for the duplexAP–Q and the waste product from the previous cycle. The gelexperiments thus show unambiguously that the aptamerdevice can be switched repeatedly between its two conforma-tions and cyclically binds and releases thrombin.

To follow the functioning of the aptamer device in realtime, FRET experiments were performed. FRET is the

nonradiative transfer of excitation energy from a “donor”fluorophore to an “acceptor” chromophore whose absorptionspectrum overlaps with the emission spectrum of the donor.The efficiency of this process is strongly dependent on thedistance between the two dyes and can be used to determinethe proximity of labeled molecules.[10] Several FRET sensorsbased on aptamers have been constructed.[7,11] Here, FRET isused to follow the conformational changes of the DNA deviceaccompanying the binding and release of thrombin. InFigure 3 the results of FRET experiments are shown in

which the strands AP3, Q5 (labeled at the 3’- and 5’-ends,respectively, see Figure 1), and R are used in the absence(Figure 3a) and presence (Figure 3b) of thrombin. Uponbinding of Q5 to AP3, the fluorophore on AP3 is quenched bythe FRET acceptor on Q5. AP3 is already partly quenchedwhen Q5 attaches to the toehold (cf. II in Figure 1); fullquenching occurs after complete binding of Q5 to AP3.Figure 3 indicates that the device can be switched from the Gquartet to the duplex form much more rapidly in the absenceof thrombin. In contrast, the removal step does not involvethrombin and displays second order kinetics with roughly thesame time constants for both situations (t= 220–240 s withoutthrombin, t= 290–350 s with thrombin).

The operation kinetics of the machine is influenced to agreat extent by the presence of the toehold section attached tothe aptamer sequence. To elucidate this effect, a shorteropening strand, Q5-S, was constructed to be complementaryto the original 15-base aptamer sequence but not to containthe complement of the toehold. In the absence of thrombin,the device rapidly undergoes a transition to the duplex formupon addition of either Q5 or Q5-S. The final fluorescencelevel for the AP3–Q5-S structure is slightly lower, since thefluorophores are closer together here than in the AP3–Q5complex. In both cases the half-lives for the transitions are onthe order of t= 5 s. The situation is very different in thepresence of thrombin. The fluorescence decay induced by theopening strand Q5 is well modeled by the superposition of a

Figure 2. Gel evidence for the operation of the DNA machine. Lanescontain: a) AP, b) AP+ thrombin (TB), c) (AP+TB)+Q, d) Q,e) Q+R, f) R, g) ((AP+TB)+Q)+R, h) (((AP+TB)+Q)+R)+Q.Pairs in brackets were incubated for 1 h before the addition of the nextcomponent. In the hybridization reactions the oligonucleotides eachhave a concentration of 1 mm. Human a-thrombin (Fluka) was addedin a protein:aptamer ratio of 10:1. Due to the higher fluorescenceintensity of the gel stain when bound to DNA duplexes, the totalamount of DNA loaded into wells c, e, g, and h was adjusted to obtaina uniform intensity for the bands without extensive broadening orsmearing. In lane g only 60% of the amount of strand AP was addedrelative to that in lane b, which explains the reduced intensity for theAP–TB complex in this lane.

Figure 3. Normalized fluorescence intensity I/I0 collected during theoperation of the DNA device, a) without thrombin, b) in the presenceof thrombin. The conformations corresponding to the different fluores-cence levels are indicated by the icons.

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second order and a first order process. The half-life for thesecond order step is t= 24 s. The half-life for the first orderstep is t= 960 s. The fast component is likely due to reactionof Q5 with thrombin-free AP strand and also due to itsbinding to and release from the toehold section (II inFigure 1). The slow first order part corresponds to the releaseof protein from the Q–AP–TB complex. When the shortopening strand Q5-S, which does not attach to the toeholdsection, is used, a much slower first order step with half-lifet= 1740 s is observed. The fits indicate that the toeholdsection increases the rate of protein release approximatelytwofold (Figure 4).

In order to directly observe the binding and release ofthrombin by the machine, measurements of the fluorescenceanisotropy r were completed (see the Experimental Sec-tion).[12] In Figure 5 r is displayed for several operation cyclesof the device. For these experiments only the aptamer strandAP3 was labeled. Fluorescence anisotropy is sensitive to theenvironment of a fluorophore and the presence of the proteinconstrains the dye's rotational degree of freedom and thusenhances the anisotropy. The change in r therefore corre-sponds to protein-binding or -release events. The opening stepconfirms the first order release kinetics with a half-life ofroughly 600 s, the binding step is second order with a half-life

t= 230 s. A thorough investigation of the kinetics of thedevice is in progress.

We have constructed a DNAmolecular machine based ona DNA aptamer that can be instructed repeatedly to bind orrelease a protein. In essence, the device functions to preciselycontrol the concentration of thrombin protein in solutionbetween a depleted and an enriched state. The combination ofthe operating principles of DNA-based nanomechanicaldevices with the binding properties of DNA aptamerssignificantly opens up design possibilities for the constructionof further functional DNA nanostructures. As aptamers canbe selected to bind to a large variety of arbitrarily chosencompounds, DNA-based nanomachines useful for carrying,binding, and releasing molecules other than thrombin couldbe easily constructed from the device reported here.

Experimental SectionThe DNA sequences for AP3, Q5, and R are shown in Figure 1: The3’-label of AP3 is Oregon Green 488 (Molecular Probes, Oregon).The 5’-label of Q5 is TAMRA (5(6)-carboxytetramethylrhodamine).Strand AP is like AP3 without the thymine spacer and the label at 3’.Unlabeled Q has the same sequence as Q5. Short Q5-S is 5’-TAMRACCAACCACACCAACC-3’; R is 5’-CAACGCCGTAAGTT-CATCTCGGTTGGTGTG-3’. All oligonucleotides were synthesizedby biomers.net, Ulm, Germany. Unless stated otherwise, chemicalswere obtained from Sigma-Aldrich, Germany, and used withoutfurther purification. The buffer used for all experiments, unlessotherwise indicated, was a modified physiological buffer consisting of20 mm Tris HCl, 140 mm NaCl, 10 mm MgCl2, and 10 mm KCl atpH 8.5.

Polyacrylamide gel electrophoresis was performed on a 15%native gel in TB buffer (89 mm Tris HCl, 89 mm boric acid) with10 mm KCl and run for 1.5 h with a field of 10 Vcm�1 at 20 8C. Gelswere stained using the nucleic acid stain SYBR gold (MolecularProbes). Thrombin is not fluorescent itself and is not stained by thedye.

For FRET experiments, fluorescence was excited with an Argonion laser (l= 488 nm) and detected with a Si photodiode through a10-nm bandpass filter centered at 514.5 nm using lock-in detection.The temperature was thermostated at 25 8C. The concentration ofstrand AP was 1 mm in all cases; other strands were added stoichio-metrically. Thrombin was added at fivefold excess over AP.

In anisotropy experiments, fluorescence was excited through aGlan–Thompson (GT) polarizer. Parallel and perpendicular compo-nents Ik, I? of the fluorescence intensity were collected through twoGTanalyzers in a T-format. Anisotropy is defined as r= (Ik�I?)/(Ik+2 I?).

Received: December 15, 2003 [Z53537]Published Online: April 28, 2004

.Keywords: aptamers · DNA structures · DNA ·molecular devices · nanostructures

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Figure 4. Fluorescence decay after the addition of a quenchersequence Q to the device in the presence of TB (circles) or without TB(squares). Every 50th data point is displayed. The continuous lines arefits to the data (see text).

Figure 5. Fluorescence anisotropy r collected during the operation ofthe aptamer device in the presence of thrombin. The signal is only sen-sitive to the binding of the device to the protein and thus directlyshows the binding and releasing of thrombin. The continuous lines arefits to the data.

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3552 � 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org Angew. Chem. Int. Ed. 2004, 43, 3550 –3553

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